Modified electrode buffers for stain-free protein detection in electrophoresis

ABSTRACT

Proteins that are electrophoretically separated in a gel are derivatized to produce fluorescent emissions by incorporating halo-substituted organic compounds into one or both of the electrode buffer solutions at the two ends of the gel. The halo-substituted compounds used are ones that bear an electric charge at the pH of the buffer solutions and gel, and the polarity of the charge on the compounds is such that the compounds migrate from the electrode buffer into the gel under the electrophoretic influence concurrently with the migration of the proteins into the gel. Once the proteins are separated and distributed within the gel and the gel is fully penetrated with the halo-substituted compounds, the gel is irradiated with ultraviolet light to induce a reaction between the halo-substituted compounds and the proteins through the tryptophan residues on the proteins, producing fluorescent reaction products.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional PatentApplication No. 61/680,587, entitled “MODIFIED ELECTRODE BUFFERS FORSTAN-FREE PROTEIN DETECTION IN ELECTROPHORESIS” and filed Aug. 7, 2012,the entire contents of which are incorporated by reference herein forall purposes.

BACKGROUND OF THE INVENTION

Gel electrophoresis, most notably in polyacrylamide gels, is one of themost common laboratory techniques for analyzing biological samples fortheir protein contents, including both identification and quantitation.Detections of proteins in gels are achieved in a variety of ways. Themost common is the use of stains such as COOMASSIE™ Brilliant Blue (BASFAktiengesellschaft, Ludwigshafen, Germany), Ponceau S (Sigma-Aldrich,St. Louis, Mo., USA), and SYPRO RUBY™ (Life Technologies). Detection canalso be achieved without stains, such as by using a stain-free techniquedisclosed by Edwards et al. in U.S. Pat. Nos. 7,569,103 B2 (Aug. 4,2009) and 8,007,646 B2 (Aug. 30, 2011). These patents describe the UVlight-induced reaction between the indole moiety of tryptophan and anyof various halo-substituted organic compounds to produce a fluorescentderivative of the protein that emits light at wavelengths in thenear-ultraviolet and visible range. The stain-free technique thusentails contacting the proteins or the gel with the halogen-containingreagent, exposing the gel to UV light once the proteins have beenseparated in bands within the gel by electrophoresis, and detecting, orforming an image of, the emissions from the proteins.

BRIEF SUMMARY OF THE INVENTION

It has now been discovered that a halo-substituted organic compound thatreacts with tryptophan residues can be distributed through a gel forreaction with proteins in the gel by imposing an electric charge on thecompound and incorporating the charged compound in one or both of theelectrode buffers in an electrophoresis system. When a biological sampleis loaded onto the gel and the electrodes that are immersed in thebuffers are energized at the appropriate polarities to causeelectrophoretic separation of the proteins in the sample to occur, thehalo-substituted compound will migrate into and through the gel byvirtue of its charge, thereby utilizing the electrophoretic principle totransfer the compound from the electrode buffer into the gel. Thepenetration of the gel with the halo-substituted compound will thusoccur concurrently with the reparatory migration of the proteins withinthe gel, avoiding any need for pre-treatment of the sample or the gel orfor post-treatment of the gel.

A method is provided herein for separating proteins contained within abiological sample by electrophoresis in a gel along a linear dimensionbetween first and second ends of the gel and detecting the proteins soseparated. The method includes the steps of: (a) loading the sample ontothe gel and electrically connecting the first and second ends of the gelwith electrodes through electrode buffers in which the electrodes areimmersed, wherein at least one of the electrode buffers has suspendedtherein a halo-substituted organic compound that reacts with tryptophanresidues upon irradiation with ultraviolet light to form fluorescentcompounds, the halo-substituted organic compound bearing an electricalcharge, (b) energizing the electrodes to opposing polarities in adirection selected to cause the proteins to distribute within the gelalong the linear dimension and to cause the halo-substituted organiccompound to migrate into the gel from the electrode buffer, and (c) withthe proteins so distributed and the halo-substituted organic compoundhaving so migrated into the gel, irradiating the gel with ultravioletlight to react tryptophan residues on the proteins with thehalo-substituted organic compound to form fluorescent derivatives of theproteins and detecting fluorescent signals emitted from said fluorescentderivatives.

In some embodiments of the method, the halo-substituted organic compoundbears a negative charge and step (b) includes energizing the electrodethat is immersed in the electrode buffer in which the halo-substitutedorganic compound is suspended as a cathode. In one such embodiment, thehalo-substituted organic compound has a molecular structure thatcontains a negative ionic moiety. In other such embodiments, thehalo-substituted organic compound is a hydrophobic compound encapsulatedin a negatively charged micelle. In one of these embodiments, themicelle is formed of sodium dodecyl sulfate.

In some embodiments of the method, the halo-substituted organic compoundbears a positive charge and step (b) includes energizing the electrodethat is immersed in the electrode buffer in which said halo-substitutedorganic compound is suspended as an anode. In some embodiments, theelectrodes at the first and second ends of the gel are immersed in thesame electrode buffer.

Also provided herein is a method of detecting proteins in anelectrophoresis gel. The method includes the steps of: loading theproteins into the electrophoresis gel; placing the electrophoresis gelbetween two electrodes; contacting the electrophoresis gel and one orboth electrodes with an electrode buffer, the electrode buffercontaining a halo-substituted organic compound; energizing theelectrodes to opposite polarities, thereby causing migration of proteinswithin the gel and transfer of the halo-substituted organic compoundfrom the electrode buffer into the electrophoresis gel; exposing theelectrophoresis gel to ultraviolet light; and detecting fluorescenceemitted from the electrophoresis gel, thereby detecting proteins in theelectrophoresis gel.

Some embodiments of the present methods also involve transferringproteins from the electrophoresis gel to a blotting membrane usingelectroblotting; exposing the blotting membrane to ultraviolet light;and detecting fluorescence emitted from the blotting membrane, therebydetecting proteins on the blotting membrane.

BRIEF DESCRIPTIONS OF THE DRAWINGS

FIG. 1 shows fluorescence emitted by proteins in Gel A of the Example.Gel A contained 0.5% trichloroethanol, which was incorporated into thegel upon pouring.

FIG. 2 shows fluorescence emitted by proteins in Gel B of the Example.No halo-substituted organic compound was incorporated into the gel uponpouring, but electrophoresis was carried out in the presence of anelectrode buffer containing 0.5% trichloroethanol.

FIG. 3 shows fluorescence emitted by proteins in Gel C of the Example.No halo-substituted organic compound was incorporated into the gel uponpouring, but electrophoresis was carried out in the presence of anelectrode buffer containing 0.5% trichloroacetic acid.

FIG. 4 shows fluorescence emitted by proteins in Gel D of the Example.No halo-substituted organic compound was incorporated into the gel uponpouring or present in the electrode buffer during electrophoresis.

DETAILED DESCRIPTION OF SELECTED EMBODIMENTS

A variety of halo-substituted organic compounds, and indeed anyhalo-substituted organic compound can be used that will enter into achemical reaction with tryptophan to form a product that fluoresces uponexposure to excitation light, can be used in the practice of thisinvention. Halo-substituted organic compounds of particular interest aretrihalo compounds, most notably those with molecular weights of 200 orless. Trihaloaliphatic alcohols, trihaloaliphatic acids,trihaloaliphatic amines, and trihaloalkanes are all useful. The halogensubstitution can be any halogen, the most convenient of which will mostlikely be chlorine and bromine. When two or more halogen atoms arepresent on a single molecule of the compound, the halogens can be thesame or a combination of different halogens. Halo-substituted organiccompounds can be used individually or in combinations, such as forexample combinations of two or three such compounds in approximatelyequal molar proportions.

The halo-substituted organic compound will bear an electrical charge,and by the terms “bear” or equivalent terms such as “bearing” or“borne,” is meant that an electrical charge is associated with oradheres to each molecule of the compound and remains so as the compoundtravels through the electrophoretic system, whether such travel is bysimple diffusion, convection, or electrophoretic migration, and ispresent at the pH of the buffer solution which the compound is suspendedor dissolved. The electric charge can be that of a moiety that is partof the molecular structure of the compound, i.e., a moiety covalentlybonded to the remainder of the compound. Examples of negatively chargedmoieties are carboxylates, phosphates, and sulfates. Specific examplesof halo-substituted compounds bearing these moieties include, but arenot limited to, trichloroacetate, tribromoacetate, iodoacetate, andtrichloropropanoate. A negatively charged compound can be introducedinto the buffer as desired, for example as a salt (e.g. sodiumtrichloroacetate) or as the conjugate acid (e.g. trichloroacetic acid).Examples of positively charged moieties are amino groups. Other examplesof both types of moieties will be readily apparent to those of skill inthe art. Alternatively, the electric charge can be imparted to thecompound by a micelle incorporating or encapsulating the compound. Ahalo-substituted organic compound that is hydrophobic and/or nonpolar incharacter, or that is uncharged (non-ionized) at the pH of the buffer,can be encapsulated in a micelle formed from detergent molecules.Examples of uncharged compounds that can be used are chloroform,bromoform, iodoform, trichloroethanol, trichloroethane, 3-bromopropanoland trichloroacetamide. Examples of detergents that form negativelycharged micelles are sodium dodecyl sulfate, sodium cholate, and sodiumdeoxycholate. An example of a detergent that forms a positively chargedmicelle is C₁₆TAB (hexadecyl trimethylammonium bromide).

The electrode buffer into which the halo-substituted organic compound isplaced will be the buffer in which the electrode of the same polaritywill be immersed, so that the compound will migrate from that bufferinto the gel when an electric potential is applied between theelectrodes. Thus, when a compound bearing a negative charge is used, thecompound will be placed in the cathode buffer, and when a compoundbearing a positive charge is used, the compound will be placed in theanode buffer. A compound bearing an appropriate charge can be placed ina single electrode buffer or compounds bearing opposite charges can beplaced in the two electrode buffers, although it will generally sufficeto place a compound in one electrode buffer only. Use of thehalo-substituted compound can be achieved without additional placementof the compound in either the sample itself prior to loading onto thegel, or in the gel prior to the commencement of electrophoresis.

In some embodiments, the anode and cathode are immersed in the sameelectrode buffer. Here, there can be two portions of electrode buffer,each covering one electrode, and the portions can be isolated from eachother (such as by the gel), so that no molecules of halo-substitutedcompound, buffering agent, or water can freely pass between them.Alternatively, the portions can be connected, allowing the free passageof molecules and ions, or both electrodes can be immersed in the sameportion of electrode buffer. Indeed, in some embodiments, the gel andother parts of the gel apparatus, as well as both electrodes, are allbathed in the same portion of electrode buffer, so that there is nosegregation among the gel and electrodes. Such embodiments can beconvenient to set up when the gel is oriented horizontally. When asingle electrode buffer is used, it can contain one or morehalo-substituted compounds of either charge, or a micelle-encapsulatedhalo-substituted compound, as discussed above.

The concentration of the halo-substituted compound in the buffer and thevolume of the buffer can vary. Excess quantities of the halo-substitutedcompound will not affect the accuracy of the fluorescent signals orproduce false fluorescent signals since only the product of the reactionbetween the halo-substituted compound and the protein (by way of thetryptophan residues) will generate a fluorescent signal. Nevertheless,for optimal results, the halo-substituted compound will be present insufficient quantity to react either with all of the tryptophan groups inthe proteins or with a proportion of the tryptophan groups that isapproximately uniform throughout the gel. In either case, it isdesirable that the signal be representative of the proteins present inthe gel rather than the degree or pattern of penetration of the gel withthe halo-substituted compound. Optimal concentrations and buffersolution volumes are readily determinable by those skilled in the art,for example by using routine experimentation on standard samples ofknown composition or by testing different concentrations and volumes onidentical samples to determine those concentrations and/or volumes abovewhich no further changes in the signal intensity and/or distribution areobserved. In most cases, optimal results will be achieved with ahalo-substituted compound at a concentration of from about 0.01% toabout 5.0% on a volume/volume basis, and with a volume of electrodebuffer (in which the compound is suspended or dissolved) of from about1.0 to about 50.0 gel volumes. When sodium dodecyl sulfate or othermicelle-forming detergent is present, its concentration may range fromabout 0.02% to about 5.0% on a weight/volume basis.

The electrode buffer(s) in which the halo-substituted compound is placedwill generally contain a buffering agent to maintain its pH at aselected level, which can vary with the sample being analyzed or theproteins sought to be detected. Examples of buffering agents are lysine,arginine, histidine, 2-(N-morpholino)-ethanesulfonic acid (MES),N-(2-acetamido)-2-aminoethanesulfonic acid (ACES),piperazine-N,N′-2-ethanesulfonic acid (PIPES),2-(N-morpholino)-2-hydroxy-propanesulfonic acid (MOPSO),N,N-bis-(hydroxyethyl)-2-aminoethanesulfonic acid (BES),3-(N-morpholino)-propanesulfonic acid (MOPS),N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid (HEPES),3-(N-tris-(hydroxymethyl)methylamino)-2-hydroxypropanesulfonic acid(TAPSO), 3-(N,N-bis[2-hydroxyethyl]amino)-2-hydroxypropanesulfonic acid(DIPSO), N-(2-hydroxyethyl)piperazine-N′-(2-hydroxypropanesulfonic acid)(HEPPSO), 4-(2-hydroxyethyl)-1-piperazine propanesulfonic acid (EPPS),N-[tris(hydroxymethyl)-methyl]glycine (Tricine),N,N-bis(2-hydroxyethyl)glycine (Bicine),[(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]-1-propanesulfonic acid(TAPS), N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonicacid (AMPSO), tris(hydroxymethyl)amino-methane (Tris), andbis[2-hydroxyethyl]iminotris-[hydroxymethyl]methane (Bis-Tris). The pHcan vary widely depending on the particular separation, but in mostcases will fall within the range of about 3 to about 10, and in certaincases from about 4.5 to about 6.5, while in other cases from about 6.5to about 9.0.

In some embodiments, a kit including one or more electrode buffers isprovided. Each electrode buffer can include a halo-substituted organiccompound and buffering agent as described above. The halo-substitutedcompound and buffering agent can be matched so that the compound bears adesired charge at or near the pKa of the buffering agent, or within theuseful pH range of the buffering agent. The kit can be tailored to aspecific electrode polarity, electrophoresis set-up, gel type, orbiological sample type, as desired.

Any electrophoresis gel can be used in the methods described herein. Forexample, the gel can be of any dimensions, have any number of lanes, andbe prepared (poured) by hand or by machine. In some embodiments, the gelcomprises polyacrylamide, which can be present at any percentage orconcentration, including at more than one concentration (e.g. instacking and resolving portions of the gel) or at a gradient ofconcentrations. The gel can also comprise a denaturing agent such assodium dodecyl sulfate (SDS), or one of the buffering agents listedabove. Other common constituents of electrophoresis gels, particularlygels used to separate complex protein samples, will be apparent to theskilled artisan. In some cases, better separation of proteins, as wellas more efficient transfer of the halo-substituted compound into thegel, can be achieved if the gel is similar in chemical composition(e.g., contains the same buffering agent) to the electrode buffer(s).

In some embodiments, the gel includes additives that allow proteins tomigrate through the gel faster and at higher applied voltages than wouldbe practicable in the absence of these additives. The additives alsoimprove separation of proteins by preventing the duplication of bands,which can result from gaps or undesired interactions between the gel andany surfaces between which it is held (see e.g. U.S. Pat. No.7,056,426). Examples of such additives include poly(vinyl alcohol),agarose, poly(vinyl pyrrolidone), poly(ethylene glycol), poly(ethyleneoxide), poly(propylene glycol), poly(propylene glycol)/poly(ethyleneglycol) copolymers, and linear polyacrylamide. Electrophoresis gelscontaining one or more of these additives are available from Bio-Radunder the name ‘TGX’.

The biological sample to be subjected to electrophoresis can be obtainedfrom any source. Examples of potential sources include cells, groups ofcells, tissues, or entire organisms, living or dead. The sample can be acell lysate, tissue homogeneate, or sample of blood, saliva, urine,cerebrospinal fluid, or other bodily fluid, among other possibilities.It will be appreciated that samples from different sources vary in thenumber, identities, and abundances of proteins that they contain, andthat many of these parameters will not be known at the time the sampleis acquired. As is well known, gel electrophoresis can be used toanalyze complex protein samples and compare these samples with eachother. Comparisons can be made between samples from different biologicalsources, such as different adult humans, humans of different ages,diseased and healthy humans, humans of different races or ethnicities orfrom different parts of the world, humans undergoing differenttreatments for diseases, humans undergoing treatments vs. humans notundergoing treatments, humans vs. non-human mammals, or any variable vs.a control. Other examples will be readily apparent to those of skill inthe art.

The number of different proteins that will most often be present in a“complex sample” as the term is used herein will be about 50 or more,often within from about 50 to about 100,000, and in many cases fromabout 100 to about 50,000. The molecular weights of these proteins canvary widely, and many such samples will have molecular weights rangingfrom those having less than twenty amino acid residues to those having1,000 or more, including as many as 5,000.

Likewise, the number of tryptophan residues among the proteins in asingle sample can range from as little as zero to as high as 5%.

Once obtained, a biological sample may require preparation before it canbe run on an electrophoresis gel and detected. Such preparation caninclude e.g. centrifuging or filtering the sample to remove tissuefragments, membranous structures, or other large contaminants;

concentrating the sample into a smaller volume by application of apressure differential; or adding chemicals to the sample such asprotease inhibitors or buffering agents. In particular, in someembodiments the sample is added to or resuspended in a buffer similar tothe electrode buffer(s) in terms of pH, salt concentration, bufferingagent, or other characteristics. This ensures that proteins of thesample will enter the electrophoresis gel and migrate within it in anefficient, reproducible manner. Other preparatory steps will be apparentto those skilled in the art. It will be appreciated that somepreparatory steps can reduce the number of proteins loaded onto theelectrophoresis gel and later reacted with the halo-substitutedcompound.

The electrophoresis gel can be run using any techniques desired, andusing any available materials or apparatus. In standard practice, thegel is contacted with the electrode buffer(s) and placed between twoelectrodes, which are energized to opposite polarities. The resultingelectric field between the electrodes drives electrophoresis and causesthe halo-substituted compound(s) to enter the gel from the electrodebuffer(s). During electrophoresis or “running”, proteins that have beenloaded in the gel migrate within the gel, away from the site of loading,and become separated from each other according to molecular weight,size, or charge. Electrophoresis can also separate proteins fromcontaminants that may have been loaded onto the gel along with theprotein sample. Such contaminants can fail to enter the gel when thepotential difference is applied, can diffuse from the gel into thesurrounding buffer, or can pass through the gel more slowly or quicklythan proteins of interest in the sample. For convenience and if desired,a molecular weight marker can be loaded into the gel along with theprotein sample, allowing the practitioner to track the positions ofproteins in the sample during or after migration.

Once the proteins and the halo-substituted compound have beendistributed through the gel by electrophoresis, the gel is irradiatedwith ultraviolet light to cause the reaction between the compound andthe tryptophan residues to occur. The irradiation intensity and durationcan vary, provided that the intensity and duration are both sufficientto cause the reaction to occur and to produce a fluorescent emissionthat can be detected and quantified. Exposure and detection can occursimultaneously. Optimum intensities and durations can be determined byroutine experimentation combined with simple observation of theresulting image. The manner of detection and quantification may varywith the type of detector used. Irradiation wavelengths within the rangeof from about 200 nm to about 400 nm, and exposure times of from aboutthirty seconds to about thirty minutes, or in many cases from about 1minute to about ten minutes, will generally provide adequate results.Irradiation can be achieved by either transillumination orepi-illumination, and detection can be achieved by imaging such as bythe use of photography, or by electronic sensors such as photodiodes,CCD detectors, or CMOS detectors. Digital results can be analyzed byconventional imaging software. Irradiation can be repeated after thecoupling reaction has occurred, for repeat detections.

The halo-substituted compounds used in the practice of the presentinvention are preferably used in the absence of any protein stains sothat the procedure is truly stain-free. By “protein stains” is meantcompounds that are color-bearing or fluorescent on their own, i.e., inthe absence of any reaction with amino acid residues, and that adhere toproteins by means other than a coupling reaction. Many such stainsexist, examples of which, as noted above, are COOMMASSIE™ BrilliantBlue, Ponceau S, and SYPRO RUBY™.

The electrophoretic procedures described herein can be used in any ofthe many forms of electrophoresis, including one-dimensional slab orcapillary electrophoresis, two-dimensional electrophoresis, andisoelectric focusing. All electrophoretic procedures can be performed inconventional ways commonly used in the laboratory and well known in theart, including loading samples onto a gel, the arrangement and use ofelectrodes and electrode buffers and the energizing of the electrodes athe appropriate polarities.

If desired, after introducing a halo-substituted organic compound intothe gel during electrophoresis and reacting the compound with proteinsof the biological sample, the proteins can be transferred out of the geland then detected. Transfer can be accomplished using electroblotting.In some embodiments, detection is then performed using an amino acidsequence within the protein that can be recognized with high affinityand specificity by a binding partner. Such a sequence is called arecognition sequence in the art, and binding partners can includeantibodies, other proteins, or small molecules. Alternatively, or inaddition, proteins can be detected outside the gel using thefluorescence of tryptophan residues that have already reacted with thehalo-substituted organic compound.

Electroblotting involves the transfer of proteins out of anelectrophoresis gel after the gel has been run, by applying an electricfield to the gel in a direction orthogonal to that used for running Thetransferred proteins are deposited onto the surface of a membrane (alsocalled a ‘blot’ or ‘blotting membrane’; typically made of nitrocelluloseor polyvinylidene fluoride (PVDF)), which can then be incubated in asolution containing the binding partner. Binding between the recognitionsequence and binding partner can be detected optically, for exampleusing fluorescence or chemiluminescence, with radioactivity, or withother means known in the art. The blotting membrane can also be exposedto UV light, and fluorescence arising from reacted tryptophan residuesin proteins on the membrane can be detected as described above or usingestablished methods.

The general method of placing one or more halo-substituted organiccompounds in electrode buffer(s) used for electrophoresis, transferringthe compounds into the gel during electrophoresis, and reacting thecompounds with tryptophan residues of proteins in the gel, can be usedto quantify the amount of protein in a biological sample or normalizethe amounts of proteins in one or more biological samples to each other.Such quantification or normalization can be performed whether proteinscontaining reacted tryptophan residues are detected in the gel, on ablotting membrane, or elsewhere. Further description of proteinquantification and normalization using stain-free methods is provided inco-pending U.S. patent application Ser. No. 13/870,710.

EXAMPLE

To demonstrate different methods of contacting proteins withhalo-substituted organic compounds in polyacrylamide gels, four gels(A-D) were loaded with protein samples and run.

Gel A was a 4-20% Criterion™ TGX™ Stain-Free Precast Gel (Bio-Rad Cat.No. 567-8094). This gel contained 0.5% trichloroethanol, which wasincorporated into the gel upon pouring, i.e. at the time of manufacture.Gels B, C, and D were 4-20% Criterion™ TGX™ Precast Gels (Bio-Rad Cat.No. 567-1094). These gels were not poured (i.e. manufactured) with anyhalo-substituted organic compound.

Each gel was inserted vertically into a Bio-Rad Criterion™ cell filledwith electrode buffer. In the case of Gels A and D, the cell was filledwith 900 mL 1× TGS (Tris/glycine/SDS) running buffer (Bio-Rad Cat. No.161-0732). In the case of Gel B, the cell was filled with buffercontaining 0.5% v/v trichloroethanol (900 mL 1× TGS running buffer plus4.5 mL trichloroethanol, Sigma-Aldrich Cat. No. T54801-100G). In thecase of Gel C, the cell was filled with buffer containing 0.5% v/vtrichloroacetic acid (900 mL 2× TGS running buffer plus 4.5 mLtrichloroacetic acid, VWR Cat. No. BDH3372-2). For each gel, the sameelectrode buffer was used to submerge both the anode and the cathode,although each electrode was covered by a separate portion of buffer.

The gels were loaded with protein samples prepared from the Bio-RadSDS-PAGE Standards (Bio-Rad Cat. No. 161-0317). First, a stock solutionof protein was prepared by diluting Bio-Rad SDS-PAGE Standards 1:10 in1× Laemmli sample buffer (50% 2× Laemmli sample buffer (Bio-Rad Cat. No.161-0737), 45% water, and 5% 2-mercaptoethanol (Bio-Rad Cat. No.161-0710)). Serial dilutions of the stock solution (1:2, 1:4, 1:8, 1:16,1:32, 1:64, 1:128, 1:256, 1:512) were then prepared in 1× Laemmli samplebuffer. The stock solution was loaded into lane 1 of each gel, anddilutions thereof were loaded into lanes 2-10. Thus, the total proteinconcentration in each lane differed by a factor of two from theconcentration in the adjacent lane(s), with the concentrationsdecreasing going from lane 1 to lane 10.

After loading, each gel was run for 30 minutes at 250 V and imaged usingthe ChemiDoc MP system. The imaging involved irradiating the gel with UVlight and detecting emitted fluorescence.

The fluorescence of Gel A (FIG. 1) demonstrates that proteins came intocontact with trichloroethanol that was incorporated into the gel uponpouring. The fluorescence of Gels B (FIG. 2) and C (FIG. 3) demonstratesthat proteins came into contact with trichloroethanol andtrichloroacetate, respectively, that entered the gel from the electrodebuffer during electrophoresis. Gel D (FIG. 4) exhibited significantlyless fluorescence than the other gels because no halo-substitutedorganic compound was incorporated into the gel upon pouring or presentin the electrode buffer. Fluorescence emitted from Gel D was likely theauto-fluorescence of aromatic amino-acid side-chains (see e.g. Roegeneret al., Analytical Chemistry 75, pp. 157-159, 2003).

In the claims appended hereto, the term “a” or “an” is intended to mean“one or more.” The term “comprise” and variations thereof such as“comprises” and “comprising,” when preceding the recitation of a step oran element, are intended to mean that the addition of further steps orelements is optional and not excluded. All patents, patent applications,and other published reference materials cited in this specification arehereby incorporated herein by reference in their entirety. Anydiscrepancy between any reference material cited herein or any prior artin general and an explicit teaching of this specification is intended tobe resolved in favor of the teaching in this specification. Thisincludes any discrepancy between an art-understood definition of a wordor phrase and a definition explicitly provided in this specification ofthe same word or phrase.

What is claimed is:
 1. A method for separating proteins contained withina biological sample by electrophoresis in a gel along a linear dimensionbetween first and second ends of said gel and detecting said proteins soseparated, said method comprising: (a) loading said sample onto said geland electrically connecting said first and second ends of said gel withelectrodes through electrode buffers in which said electrodes areimmersed, wherein at least one of said electrode buffers has suspendedtherein a halo-substituted organic compound that reacts with tryptophanresidues upon irradiation with ultraviolet light to form fluorescentcompounds, said halo-substituted organic compound bearing an electricalcharge, (b) energizing said electrodes to opposing polarities in adirection selected to cause said proteins to distribute within said gelalong said linear dimension and to cause said halo-substituted organiccompound to migrate into said gel from said electrode buffer, and (c)with said proteins so distributed and said halo-substituted organiccompound having so migrated into said gel, irradiating said gel withultraviolet light to react tryptophan residues on said proteins withsaid halo-substituted organic compound to form fluorescent derivativesof said proteins and detecting fluorescent signals emitted from saidfluorescent derivatives.
 2. The method of claim 1 wherein saidhalo-substituted organic compound bears a negative charge and step (b)comprises energizing said electrode that is immersed in said electrodebuffer in which said halo-substituted organic compound is suspended as acathode.
 3. The method of claim 1 wherein said halo-substituted organiccompound bears a positive charge and step (b) comprises energizing saidelectrode that is immersed in said electrode buffer in which saidhalo-substituted organic compound is suspended as an anode.
 4. Themethod of claim 2 wherein said halo-substituted organic compound has amolecular structure that contains a negative ionic moiety.
 5. The methodof claim 2 wherein said halo-substituted organic compound is ahydrophobic compound encapsulated in a negatively charged micelle. 6.The method of claim 5 wherein said micelle is formed of sodium dodecylsulfate.